Reducing morbidity in surgical resections: Third-harmonic generation microscopy a

描述（由申请人提供）：神经意外损伤是肿瘤手术及其他手术中发病的主要原因之一。在手术过程中，神经纤维很难与周围的组织区分开来，这使得外科医生很难仔细地在周围工作并避免损伤。总的来说，本研究的目标是建立三次谐波生成（THG）显微镜，作为术中观察神经纤维的一种高灵敏度方法。我们之前已经证明THG提供了一种高灵敏度的方法来成像啮齿动物中枢神经系统中的髓磷脂，并将其用于脊髓髓磷脂的体内成像。我们最近收集的初步数据表明，这种方法也可以突出周围神经。最终，这种成像方式可以让外科医生看到神经纤维，然后在它们附近仔细工作，从而防止与神经损伤相关的手术并发症。我们确定了完成这项工作所必需的三个具体目标。首先，我们在初步数据的基础上，确定了一套最佳的激光和成像参数，以THG成像显示髓鞘周围神经束。其次，我们通过比较采用和不采用这种新型成像方式的啮齿动物前列腺切除术的结果来评估THG成像对节省神经的有效性。在本研究中，我们进一步建立在先前的结果基础上，不仅研究了THG可视化时神经的幸免程度，而且还研究了双光子激发荧光（2PEF）显微镜成像时肿瘤的完全提取程度。至关重要的是，THG成像所需的激光和成像系统与2PEF成像完全兼容，允许这两种模式协同工作，帮助外科医生最大限度地提取肿瘤并最大限度地减少神经损伤。综上所述，这两个目标将建立我们的神经成像方法的实用性，但在手术中使用

DESCRIPTION (provided by applicant): Unintentional injury to nerves is one of the primary causes of morbidity during surgical resection of tumors as well as in other surgeries. Nerve fibers are difficult to distinguish from the surrounding tissues during surgery, making it difficul for surgeons to carefully work around and avoid their injury. Broadly speaking, the goal of the proposed work is to establish third-harmonic generation (THG) microscopy as an approach to visualize, with high sensitivity, nerve fibers intraoperatively. We have previously shown that THG provides a high-sensitivity approach to imaging myelin in the central nervous system of rodents and have used this for in vivo imaging of myelin in the spinal cord. We recently collected preliminary data that shows this approach can highlight peripheral nerves as well. Ultimately, this imaging modality could allow the surgeon to visualize nerve fibers and then work carefully near them, thereby preventing surgical complications associated with nerve injury. We identify three Specific Aims necessary to complete this work. First, we build on our preliminary data and identify the best set of laser and imaging parameters to visualize myelinated peripheral nerve bundles with THG imaging. Second, we assess the effectiveness of THG imaging to spare nerves by comparing the outcome of prostate resection surgeries in rodents done with and without this novel imaging modality. In this Aim, we further build on previous results and examine not just how well nerves are spared when visualized with THG, but also how completely tumors are extracted when they are imaged with two-photon excited fluorescence (2PEF) microscopy. Critically, the laser and imaging systems necessary for THG imaging are completely compatible with 2PEF imaging, allowing these two modalities to work in concert to help a surgeon both maximize tumor extraction and minimize nerve damage. Taken together, these two aims will establish the utility of our approach to nerve imaging, but use in an operating room environment will clearly require development of a less-unwieldy instrument than our current microscopes. Toward this end, in the third Aim, we design an endoscope that is optimized for THG (as well as 2PEF) imaging and could be used in laparoscopic surgeries (such as many prostate cancer resections) in humans. Taken together, this work will establish the utility of THG as a viable imaging modality for use in surgery and will develop the first-generation instrument that could be used in an operating room. If successful, this work could dramatically reduce the post-surgical morbidity of resection surgeries - especially in cases where critical nerves are close to the resection site, such as in the prostate - and therefore improve patient outcomes.